Composition comprising vicenin-2 having a beneficial effect on neurological and/or cognitive function

ABSTRACT

The invention relates to an active ingredient and/or to a composition having beneficial effect on neurological and cognitive functions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Stage application ofPCT/EP2012/074010 filed 29 Nov. 2012, which claims priority to EuropeanPatent Application 11009430.7 filed 29 Nov. 2011, European PatentApplication 12163578.3 filed 10 Apr. 2012, U.S. Provisional PatentApplication 61/564,374 filed 29 Nov. 2011, and U.S. Provisional PatentApplication 61/622,260 filed 10 Apr. 2012, the entire disclosures ofwhich are hereby incorporated by reference.

TECHNICAL FIELD

The invention relates to a food, a dietary supplement and a drugcomposition comprising as an active ingredient vicenin 2 or abiologically active analogue thereof, and especially its beneficialeffects to maintain and/or improve neurological and brain function andto prevent, delay onset, control and/or treat a cognitive dysfunction,condition, disorder or disease, e.g. dementia such as Alzheimer disease.

BACKGROUND OF THE INVENTION

Maintenance or improvement of neurological and brain functions isimportant to vitality and well being throughout all stages of life butparticularly important for elderly people. The efficiency of eachperson's neurological and brain functions can make all the difference indaily and overall health. The ability of cognitive functions, includingmemory, attention, concentration, alertness, mental flexibility andspeed, learning, intelligence, language, problem solving capacity,consciousness, coping with psychological stress or tension, motivation,mobility, decision making and reaction time as well as emotions likeanxiety and mood swings affect social and economical status and theoverall quality of life. The wish to extend cognitive capacity longer,frame a lifelong challenge, as everybody is effected by aging. Inaddition, there is an increasing number of individuals, who developneurodegenerative diseases, like dementia or Alzheimer disease. [1,2]

Dementia can be divided into three types, referring to their cause ofdevelopment. The first type is called vascular dementia, caused byreduced circulation; the second type is called secondary dementia,generated as a side effect of e.g. hormonal disorders and the third one,is Alzheimer. Alzheimer disease is the most common form of dementia,forming up to 60% of the cases. There is no cure for the disease, whichworsens as it progresses, and eventually leads to death.

Alzheimer is a devastating neurological disorder that affects more than37 million people worldwide. The economic burden of Alzheimer's diseaseis massive and currently approved drugs do not prevent or reverse thedisease and provide only modest symptomatic benefits. [3] Thepathogenesis of Alzheimer disease is not completely understood. Majorcharacteristics of Alzheimer's disease (AD) are synaptic loss,cholinergic dysfunction, and abnormal protein depositions in the brain.A combination of several causes may lead to the development of plaquesin the brain, which decrease the efficiency of neuronal communicationleading to death of neurons. Scientifically these plaques are called“beta amyloid peptides” or “Tau-proteins”. [3,4] Recent studiesconfirmed that a genetic disposition may be involved and/or infectionmay contribute to the formation of plaques and the development ofAlzheimer disease. The decline in neurological and brain functions islinked to the reduced number of neurons, which lead to a reducedsynthesis of the neurotransmitter acetylcholine and glutamine. Theincrease of neurotransmitter concentration, by inhibiting its metabolismis a possibility to promote beneficially the neurological and brainfunction or to reduce the development of dementia like Alzheimerdisease. [4]

Not many drugs are approved for the treatment of dementia such asAlzheimer. Four approved drugs target the increase of neurotransmitterconcentration. Recent drug developments and areas of clinical researchfocus on treating the cause of the disease by reduction of amyloid betalevels. Immunotherapy or vaccinations with anti-amyloid antibodies arebeing investigated. Another substance, called PBT2 binds metals whichare necessary for building of protein structures forming the plaques.Other proteins may have the ability to cut amyloid beta into smallpieces, which can be eliminated. In addition, gab junction antagonistsare being investigated. They block confused communication between cellsto promote a healthy neurological and brain functionality. [3,4] As of2012, more than 1000 clinical trials have been or are being conducted tofind ways to treat Alzheimer disease. [1,1] Despite considerable effortsby academic researchers and pharmaceutical and food industry, thedevelopment of novel ingredients for the maintenance and improvement ofneurological and brain function and to prevent the development and toslow down the progression of dementia and Alzheimer disease has beendragging and did not lead to many innovations. Thus, there is a need todevelop drugs, functional foods and dietary supplement ingredients whichmay contribute to the maintenance and improvement of neurological andbrain function and which may prevent the development of cognitivedisorders or slow down the progression of dementia and Alzheimerdisease.

The object for the present invention was to provide novel activeingredients suitable as functional food, dietary supplement ingredientsand drugs, which are able to maintain and improve neurological and brainfunction and to prevent, delay onset, control and treat cognitivedysfunctions, conditions, disorders or diseases, in particular dementiasuch as Alzheimer disease.

SUMMARY OF THE INVENTION

In accordance with the present invention it has been surprisingly foundthat the active ingredient vicenin 2 or biologically active analoguesthereof and compositions comprising the same have beneficial effects oncognitive functions including memory, attention, concentration,alertness, mental flexibility and/or speed, learning, intelligence,language, problem solving capacity, consciousness, coping withpsychological stress or tension, motivation, mobility, decision makingcapacity and reaction time as well as emotions like anxiety and moodswings.

Thus, the invention describes a new neurological and cognitive activeagent which helps to maintain and/or improve neurological and brainfunction and/or to prevent and/or improve cognitive changes and toprevent and/or treat cognitive disorders like dementia. Preferably,dementia is Alzheimer disease. The cognitive changes may be stress orage related cognitive changes. These changes may be mild.

In particular, the invention relates to vicenin 2 or a biologicallyactive analogue thereof as an active ingredient for use in maintainingand/or improving neurological and/or brain function and/or preventing,delaying onset, controlling and/or treating a neurological dysfunction,condition, disorder or disease.

Preferably, the neurological condition is associated with impairedneurological and/or brain function. In other words, preferably, themaintaining and/or improving neurological and/or brain function may beassociated with a neurological dysfunction, condition, disorder ordisease.

Thus, the present invention may relate to vicenin 2 or a biologicallyactive analogue thereof as an active ingredient for use in preventing,delaying onset, controlling and/or treating a neurological dysfunction,disorder, disease or condition, which is associated with impairedneurological and/or brain function.

In a further embodiment, the invention relates to a use of vicenin 2 ora biologically active analogue thereof as an active ingredient formaintaining and/or improving neurological and/or brain function. Thisembodiment relates to a use of vicenin 2 or a biologically activeanalogue thereof for healthy individuals, who wish to e.g. enhancecognitive performance and/or improve well-being at stress situations.

In a further embodiment, the invention relates to vicenin 2 or abiologically analogue thereof as an active ingredient for use inimproving cognitive functions, memory, attention, concentration,alertness, mental flexibility and/or speed, learning, intelligence,language, problem solving capacity, consciousness, coping withpsychological stress or tension, motivation, mobility, decision makingcapacity, reaction time, anxiety and/or mood swings in a patientsuffering from a neurological dysfunction, disorder or disease.

Preferably, the neurological dysfunction, condition, disorder or diseaseis an age or stress related neurological dysfunction, anxiety, acognitive disorder, depression or dementia, wherein dementia ispreferably Alzheimer disease. Furthermore, the active ingredient ispreferably derived from a plant and the active ingredient is a plantpreparation enriched for the active ingredient. Preferably, the plant isselected from a group consisting of a Anethum, Perilla, Urtica,Passiflora, Camelia, Cayaponia, Colocasia, Cydonia, Desmodium, Hordeum,Origanum, Ocimum, Jatropha, Parkinsonia, Peperomia, Piheranthos,Centaurea, Indigo, Bomba, Lychnophera, Asplenium, Chinotto, Citrus,Viola, Trigonella, Rosemary, Peppermint, Thyme, Basil, Sage, Oregano,Lavandula, Nipponanthemum, Abrus, Viola, Santalum, Oryza, Scleropyrum,Tulsi, Centaurea, Indigofera, Bombax, Glinus, Lychnophora and otherspecies belonging to the Lamiacea, Labiatae, and Urticaceae, Rosales orMalpighiales or a combination of said plants.

The plant preparation may be selected from the group consisting of aleaf preparation, a fruit preparation, a seed preparation, a stempreparation, a flower preparation, a bud preparation, a root preparationor a mixture of different parts of the plant.

In one embodiment, the active ingredient is an isolated vicenin 2 or abiologically active analogue thereof obtained by isolation or chemicalsynthesis.

In a preferred aspect of the present invention, the neurological and/orbrain function is selected from the group consisting of memory,attention, concentration, alertness, mental flexibility and/or speed,learning, intelligence, language skills, problem solving capacity,consciousness, coping with psychological stress or tension, motivation,mobility, decision making capacity, reaction time and regulation ofemotions. The emotions may be e.g. anxiety and/or mood swings. Thesefunctions may be impaired in association with a cognitive disorder suchas neurodegenerative disorder or dementia.

The active ingredient may be comprised in a preferred embodiment in acomposition such as a food product, dietary supplement or medicament,preferably wherein the medicament 1 to comprises a pharmaceuticallyacceptable carrier. In these aspects, the concentration of the activeingredient is from 0.1 μg to 500 μg, preferably from 2.5 μg to 50 μg,preferably from 5 μg to 15 μg or 12 μg to 30 μg, most preferably about24 μg.

Most preferably, the active ingredient is administered at a dose of0.1-0.5 μg/kg, 0.17-0.42 μg/kg or 0.07-0.3 μg/kg.

Furthermore, in a preferred embodiment the active ingredient may becomprised in globules, pellets, powder formulations, tablets, capsules,stick formulations, sachet formulations or a fluid. The fluid may be ina bottle with a dropper.

Preferably, the composition is substantially free of other plantflavonoids derived from the same plant or other plant extracts or freeof flavonoids of other plants.

In another preferred embodiment, the composition comprises one or morefurther agents capable of maintaining and/or improving neurologicaland/or brain function and/or preventing, delaying onset, controllingand/or treating a neurological dysfunction, condition, disorder ordisease as defined in any one of the preceding claims. This agent may bea lipid, a lipid containing omega-3-fatty acid, a physiologically activefatty acid, an antioxidant, an anti-inflammatory agent, a bulking agent,an immune system modulatory agent or a vaccine, an antibody, ametal-protein interaction attenuation agent, a plant preparation,curcumin, coenzym Q10, L-carnitine, zinc, epigallocatechingallate,thymol, p-cymere, vinpocetine, hyperzine A, phosphatidylserine, avitamin, alpha liponic acid, a TNF alpha inhibitor, a flavonoid, ananthocyanidin, a biflavonoid, a flavon, a flavonglycoside or acarboxylic acid.

Preferably, the plant preparation is selected from one or more extractsfrom a group consisting of an extract of Ginkgo biloba, Hypericumperforatum, Hyperzia serrata, Galanthus nivalis, Salvia officinalis,Panex ginseng, Lippia citriodora, Melissa officinalis, Passifloraincarnate, Passiflora edulis, Bacopa monniera, Zingiber officinalis,Leucojum aestrum, Concolulus pluricaulis and Centella asiatica, Emblicaofficinalis, Coptidis Rhizoma, Salvia triloba, Piper nigrum, Trigonellafoenum-graecum, Cimicifuga racemosa, Salvia miltiorrhiza, Rhodiolarosea, Habranthus jamesonii, Phycella herbertiana, Rhodophialamendocina, Zephyranthes filifolia, Stephania pierrei, Kaempferaparviflora, Stephania venosa, Crocus sativus, Salvia species, Bacopamonnieri, Centella asiatica, Ptychopetalum olacoides, Withaniasomnifera, Coptis chinensis, Mangifera indica, Polygala caudata,Polygala tenuifolia, Halenia elliptica, Evolvilus alsinoides, Celastruspaniculatus, Clitoria ternatea, Curcuma longa, Acorus calamus,Terminalia chebula, Lycoris radiata, Magnolia officinalis, Biotaorientalis, Codonopsis pilosula, Evodia rutaecarpa, Polygonummultiflorum, Aspalathus linearis, Cyclopia species, Adansonia digitata,Sclerocarya birrea, Actinidia chinensis, Matricaria recutita orcombinations thereof. In a more preferred embodiment, the plantpreparation is a preparation of Melissa officinalis, Rhodiole rosea,Magnifera indica or a Cyclopie species or any combination thereof.

In a preferred embodiment, the agent capable of maintaining and/orimproving neurological and/or brain function and/or preventing, delayingonset, controlling and/or treating a neurological dysfunction,condition, disorder or disease is an acetylcholinesterase inhibitor, aNMDA (N-methyl-D-aspartate) receptor inhibitor, a non steroidalanti-rheumatic agent, a neuroleptic agent, a tricyclic antidepressant,an anti-psychotic agent, a gab junction inhibitor, a non selectivemonoaminooxidase inhibitor, an ABCC1 Transporter, an ADAM 10 protein,methylthioninium chloride, an antibiotic agent, an antiviral agent, agamma secretase inhibitor, a beta secretase inhibitor, an angiotensinreceptor antagonist (AT1 antagonist), a cannabinoid, allopregnanolone oran insulin sensitizer.

In a further embodiment the present invention relates to a process ofproducing a preparation of the active ingredient as defined abovecomprising the steps of:

-   a) selection of the raw plant material,-   b) preparation of the raw plant material,-   c) applying an extraction process using solvent extraction and/or    filtration techniques, preferably followed by concentration and/or    spray drying of the liquid extract into a powder,-   d) determining the concentration of the active ingredient; and-   e) selecting the preparation comprising the active ingredient at a    concentration of at least 0.01%.

Preferably, the process comprises an extraction process using aqueoussolvent extraction, more preferably water extraction.

Preferably, the solvent is water. In a more preferred embodiment, theextraction is carried out at a temperature of 90° C. to 125° C. and/orthe temperature of the solvent is 55° C. or above, preferably 70° C. orabove.

Preferably, the plant is selected from one or more plants of the groupconsisting of Anethum, Perilla, Urtica, Passiflora, Camelia, Cayaponia,Colocasia, Cydonia, Desmodium, Hordeum, Origanum, Ocimum, Jatropha,Parkinsonia, Peperomia, Piheranthos, Centaurea, Indigo, Bomba,Lychnophera, Asplenium, Chinotto, Citrus, Viola, Trigonella, Rosemary,Peppermint, Thyme, Basil, Sage, Oregano, Lavandula, Nipponanthemum,Abrus, Viola, Santalum, Oryza, Scleropyrum, Tulsi, Centaurea,Indigofera, Bombax, Glinus, Lychnophora and other species belonging tothe Lamiacea, Labiatae, Urticaceae, Rosales or Malpighiales orcombinations thereof.

In addition, the invention relates to a preparation of the activeingredient obtainable by the above described process. The preparationmay be used as an active ingredient for use in maintaining and/orimproving neurological and/or brain function and/or for preventing,delaying onset, controlling and/or treating a neurological dysfunction,condition, disorder or disease as defined in any one of the precedingclaims.

Preferably, the preparation comprises the active ingredient at aconcentration of at least, 0.02%, more preferably, at a concentration ofat least 0.1%, 0.15%, 0.2% or most preferably at a concentration of atleast 0.2%, 0.25% or 0.3%.

In some cases, the preparation may comprise the active ingredient at aconcentration of at least 0.001%, more preferably, at a concentration ofat least 0.002%, 0.005%, 0.008% or preferably at a concentration of atleast 0.010%, 0.015% or 0.02%.

In some cases, the preparation may comprise the active ingredient at aconcentration of at least 0.2%, more preferably, at a concentration ofat least 0.5%, 1.5%, 3.0% or most preferably at a concentration of atleast 2.0%, 3.0% or 5.0%.

Detailed descriptions of conventional extraction methods, such as thoseemployed herein can be found in the literature, for example in the book“Industrial Scale Natural Products Extraction”, published in 2011 byWiley-VCH.

The analytical method to determine the vicenin 2 concentration may bee.g. a chromatographic method called high performance liquidchromatography. High performance liquid chromatography (HPLC) is one ofthe most popular techniques of analytical chemistry. A UV detector isused, detecting at 320 nm. For determining the concentration of vicenin2, a vicenin 2 reference or its derivates may be used as referencematerial. For example, apigenin may be used as reference substance andthe vicenin 2 content is calculated via the difference in moleculeweight and the slope and intercept of the calibration curve forapigenin.

In the process, the raw plant material is preferably Perilla species,more preferably Perilla frutescens Britton var. crispa or var. acutaKudo. The raw plant material is preferably prepared by drying, cuttingand/or milling. Extraction can be preferably done with a raw materialwhich particle size is reduced to lower than 2 mm². The solvent may be,preferably, water, methanol, ethanol, propanol, isopropanol, ethylacetate, hexane, chloroform or dichloromethane. Most preferably anaqueous solvent, preferably water, is used since glycosides, likevicenin 2, which have hygroscopic properties, are extracted by hot watermore efficiently than alcohol. Moreover, some volatile, allergeniccompounds like Perillaldehyde, Methyleugenol and Myristicin, which mayoccur in targeted species containing vicenin 2, can be eliminated by hotwater.

Ratio of raw material to solvent is preferably between 1:100 to 20:100and more preferably 2:100 to 10:100.

Extraction can be preferably done by room temperature to up to 150° C.Extraction is more preferably carried out from 90° C. to 125° C. In afurther preferred embodiment, heat with additional pressure can be used.

In a preferred embodiment the extraction time is 10 min to 2 hours, morepreferably from 20 min to 50 min.

Preferably, the plant for the process is selected from one or moreplants from the group consisting of Anethum, Perilla, Urtica,Passiflora, Camelia, Cayaponia, Colocasia, Cydonia, Desmodium, Hordeum,Origanum, Ocimum, Jatropha, Parkinsonia, Peperomia, Piheranthos,Centaurea, Indigo, Bomba, Lychnophera, Asplenium, Chinotto, Citrus,Viola, Trigonella, Rosemary, Peppermint, Thyme, Basil, Sage, Oregano,Lavandula, Nipponanthemum, Abrus, Viola, Santalum, Oryza, Scleropyrum,Tulsi, Centaurea, Indigofera, Bombax, Glinus, Lychnophora and otherspecies belonging to Lamiaceae, Labiatae, Urticaceae, Rosales orMalpighiales or combinations thereof.

The process of the invention provides an extract with a higherconcentration of the active ingredient as the prior art processes due toconcentration of the active ingredient. The higher concentration isachieved using state of the art extraction equipment, which allows anoptimal interaction between the raw material and the extractionsolvents. All critical process steps, for example temperature, arecontrolled in process at any times and adaptations are continuouslypossible to ensure highest yield for the active ingredient. Due to theselection of the extraction solvents combined with optimized physicalconditions it is possible to diminish unwanted substances which mayoccur naturally in the different raw materials.

In one embodiment, the invention provides a method for maintainingand/or improving neurological and/or brain function and/or forpreventing, delaying onset, controlling and/or treating a neurologicaldysfunction, condition, disorder or disease in a subject in need thereofcomprising administering an active ingredient as defined above, thecomposition as defined above or the preparation as defined above.Preferably, the neurological dysfunction, condition, disorder or diseaseis an age or stress related neurological dysfunction, anxiety, acognitive disorder, depression or dementia, wherein preferably thedementia is Alzheimer disease.

In a preferred embodiment, the neurological and/or brain function isselected from the group consisting of memory, attention, concentration,alertness, mental flexibility and/or speed, learning, intelligence,language, problem solving capacity, consciousness, coping withpsychological stress or tension, motivation, mobility, decision makingcapacity, reaction time and regulation of emotions.

In addition, the present invention provides a kit comprising an activeingredient as defined above, the composition as defined above or thepreparation as defined above and instructions for administering saidcomposition. Preferably, the kit is for use in maintaining and/orimproving neurological and/or brain function and/or preventing, delayingonset, controlling and/or treating a neurological dysfunction,condition, disorder or disease.

The present invention further provides a composition comprising theactive ingredient as defined above, the preparation as defined above orthe composition as defined above for use in preventing, delaying onset,controlling and/or treating a disorder or disease associated withautonomous neuronal death by blocking the gap junction hemichannel ofactivated microglia and reducing their glutamate release. Preferably,said disorder or disease is a neurodegenerative disorder, such asAlzheimer disease, Parkinson disease, Huntington disease, multiplesclerosis, amylotrophic lateral sclerosis (ALS), viral encephalitis orAIDS. Preferably, said composition comprises or is derived from a plantpreparation comprising the active ingredient. More preferably, the plantpreparation is derived from a plant and/or plant preparation as definedabove. In this embodiment the composition is preferably Perilla extract.

In a further embodiment, the present invention relates to a compositioncomprising the active ingredient, i.e. vicenin 2 or biologically activeanalogue as thereof as defined above or the preparation as definedabove, or the composition as defined above for use in preventing,delaying onset, controlling and/or treating a condition associated withglutamate and tumor necrosis factor α released by activated microgliainducing excitotoxic neuronal death. In this embodiment, the compositionis preferably Perilla extract.

Furthermore, the present invention relates to a composition comprisingthe active ingredient having one or more effects, preferably more thanone, more preferably more than two, more than three, more than four ormore than five effects, selected from the group consisting of reversibleacetylcholinesterase inhibition, blockade of gap junction hemichannels,neuroleptic effect, anti-depressive effect, enkephalin like effect andreduction in TNF alpha for use in maintaining and/or improvingneurological and/or brain function and/or preventing, delaying onset,controlling and/or treating neurological dysfunction, condition,disorder or disease. Preferably, said condition is associated withimpaired neurological and/or brain function.

In a further embodiment, the invention relates to a vicenin 2 or afunctionally active derivative thereof as an active ingredient for usein maintaining and/or improving neurological and/or brain functionand/or preventing, delaying onset, controlling and/or treating aneurological dysfunction, condition, disorder or disease, wherein theactive ingredient is not derived from Perilla.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Acute effects of vicenin 2 (AUC-V) on the cortical networkactivity in vitro. Plotted are the spike rate changes for treatment of 9accumulating concentrations in the range of 10 pg/ml to 300 μl/ml(mean±standard error, n=17; Student's paired t-test: * p≦0.05: **p≦0.01; *** p≦0.001).

FIG. 2: Acute effects of Perilla leaf extract (AUC-P) on the corticalnetwork activity in vitro. Plotted are the spike rate changes fortreatment of 9 accumulating concentrations in the range of 10 ng/ml to 1mg/ml (mean±standard error, n=5; Student's paired t-test: * p≦0.05; **p≦0.01; *** p≦0.001).

FIG. 3: A dose-depending effect of a plant extract comprising vicenin 2was shown to reduce TNFα. TNF alpha secretion [pg/ml] afterco-incubation with LPS and Perilla extract of different concentration (1μg/ml, 2.5 μg/ml and 50 μg/ml) against 450 μg/ml a benchmark blend,containing e.g. Boswellia extract, Omega 3 fatty acids, ALA andCurcumin.

DETAILED DESCRIPTION OF THE INVENTION

The inventors surprisingly found an agent having beneficial effects onneurological function and brain health, like cognitive functions,including memory, attention, concentration, alertness, mentalflexibility and/or speed, learning, intelligence, language, problemsolving capacity, consciousness, coping with psychological stress ortension, motivation, mobility, decision making capacity and reactiontime as well as emotions like anxiety and mood swings.

In particular, the inventors surprisingly found that vicenin 2 orfunctionally active derivatives, i.e. biologically active analogues,thereof have beneficial effects to maintain and improve neurological andbrain function and to prevent and improve age or stress relatedcognitive changes, which may be mild, and to prevent and treat cognitivedisorders, for example dementia, in particular Alzheimer disease. Thisefficacy is very beneficial for cognitive functions, including memory,attention, concentration, alertness, mental flexibility and/or speed,learning, intelligence, language skills, problem solving capacity,consciousness, coping with psychological stress or tension, motivation,mobility, decision making and reaction time as well as emotions likeanxiety and mood swings.

Although Alzheimer disease develops differently for every individual,there are many common symptoms. Early symptoms are decrease in memory,concentration and alertness, escalating into confusion, mood swings andemotional difficulties, loss in orientation, language problems and lossof long-term memory. Even early symptoms lead to a disconnection tofamily and society, which triggers mood swings, including aggression aswell as anxiety. In the advanced status of Alzheimer disease bodyfunctions decrease ultimately leading to death. [4]

The pathogenesis of Alzheimer disease is not completely understood and acombination of several causes may lead to the development of plaques inthe brain, which decrease the efficiency of neuronal communicationleading to die off of neurons. Scientifically these plaques are called“beta amyloid peptides” or “Tau-proteines”. Recent studies confirmedthat a genetic disposition may be involved and/or infection maycontribute to the formation of plaques and the development of Alzheimerdisease. [3,4]

First-line therapy for all forms of dementia, including Alzheimer, isintellectual activities, such as reading, playing board games,completing crossword puzzles, playing musical instruments, or regularsocial interaction. Life experiences result in more efficient neuralfunctioning providing the individual a cognitive reserve that delays theonset of dementia manifestations. Physical activity may also beassociated with a reduced risk of dementia and the slowdown inprogression. Psychosocial therapies to reduce daily stress and anxietyare as important as dietary modifications to increase the intake ofantioxidants. [4]

Several dietary supplement products are available to support brainhealth. Most of them are linked to antioxidant and anti-aging efficacy,for example, long chain omega 3 fatty acids, an physiological activefatty acid, eicosapenteanoic acid EPA, docosahexeanoic acid DHA, Ginkgobiloba extract, Curcumin or other polyphenols, catechins, flavonoids orphenolic carboxylic acid based plant preparations, including TCMs.Choline and Uridine monophosphate are also used as well as Vitamins,like folic acid and the pyridoxine (B6) and B12, vitamin C, vitamin Eand selenium. The functional food products for these areas can also bedefined as medical food which use and intake is to be recommended andsupervised by a medical doctor. The medical food market segment is adeveloping market segment.

The invention provides a new important possibility of application forbrain health, to maintain and improve neurological and brain functionand to prevent and improve age or stress related associated cognitivechanges, which may be even mild, and to prevent and treat cognitivedisorders, dementia and Alzheimer disease.

Surprisingly, as shown by the Examples the active ingredient of theinvention and the composition comprising the active ingredientdemonstrate a plurality of modes of action beneficial for neurologicaland/or brain function. Thus, the composition comprising the activeingredient can be used for treating an individual suffering from severalconditions. These may include conditions associated with reversibleacetylcholinesterase inhibition, blockade of gap junction hemichannelsand reduction in TNF alpha. Furthermore, the active ingredient as wellas the composition comprising the same show neuroleptic effect andanti-depressive effects. Therefore, the active ingredient andcompositions comprising the same result in a higher success rate in thetreatment of an individual suffering from a neurological dysfunction,condition, disorder or disease. The disorder may be a neurodegenerativedisorder, preferably Alzheimer disease, Parkinson disease, Huntingtondisease, multiple sclerosis, amylotrophic lateral sclerosis (ALS),encephalitis or AIDS.

In addition to above, the active ingredient may be used in treatment ofindividuals suffering from more than one dysfunctions, conditions,diseases or disorders. These may include e.g. dementia, in particularAlzheimer disease, a disorder associated to neuronal cell death orinflammation.

A further advantage of the composition of the invention comprising theactive ingredient is that it has a plurality of therapeutic properties;i.e. the active ingredient or composition comprising the same has one ormore of the following properties (i) an anti-inflammatory effect, (ii)anti-nociceptic effect, (iii) sedative effect, (iv) anxiolytic effect,(v) anti-cancer effect, (vi) immuno-modulation inducing effect and/or(vii) a beneficial effect on cognitive behavior and/or mood.

DEFINITIONS

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “an antibody”, is understood to representone or more antibodies. As such, the terms “a” (or “an”), “one or more”,and “at least one” can be used interchangeably herein.

By an “isolated” ingredient, variant, or derivative thereof is intendedan agent that is not in its natural milieu. No particular level ofpurification is required. For example, an isolated active ingredient canbe removed from its native or natural environment. Syntheticallyproduced active ingredients are considered “isolated” for purpose of theinvention, as are native or synthetic active ingredients which have beenseparated, fractionated, or partially or substantially purified by anysuitable technique.

By “subject” or “individual” or “animal” or “patient” or “mammal”, ismeant any subject, particularly a mammalian subject, e.g., a humanpatient, for whom diagnosis, prognosis, prevention, or therapy isdesired.

“Maintaining a healthy brain” according to the invention can beunderstood as maintaining a normal neurological and brain function,wherein the normal cognitive function encompasses several domains,including memory, attention, concentration, alertness, mentalflexibility and/or speed, learning, intelligence, language, problemsolving capacity, consciousness, coping with psychological stress ortension, motivation, mobility, decision making and reaction time as wellas emotions like anxiety and mood swings.

“Cognitive changes” relate to changes, for example, in memory,attention, concentration, alertness, mental flexibility and/or speed,learning, intelligence, language, problem solving capacity,consciousness, coping with psychological stress or tension, motivation,mobility, decision making and reaction time as well as emotions likeanxiety and mood swings. These changes may be age or stress related.Furthermore, the changes may be mild, moderate or strong.

“A neurological condition” may refer to a state of an individual havingone or more impaired neurological and/or cognitive functions. This stateis milder than a disorder or disease, and may be caused, for example, byage or stress.

“Prevention of dementia, in particular Alzheimer disease, or improvementof the status of dementia, in particular Alzheimer disease” according tothe invention can be understood as that the active ingredient hasbeneficial physiological effects to prevent or improve neurologicalfunction, cognitive functions, including memory, attention,concentration, alertness, mental flexibility and/or speed, learning,intelligence, language, problem solving capacity, consciousness, copingwith psychological stress or tension, motivation, mobility, decisionmaking and reaction time as well as emotions like anxiety and moodswings.

“Delaying onset of a dysfunction, condition, disorder or disease” can beunderstood as shifting the beginning of the dysfunction, condition,disorder or disease to a later time point.

“Controlling a dysfunction, condition, disorder or disease” meanskeeping or stabilizing a dysfunction, condition, disorder or disease ata level, which does not worsen dramatically.

“Dementia” means in this context a significant loss of intellectualabilities such as memory capacity, severe enough to interfere withsocial or occupational functioning.

“Alzheimer disease” means in this context a progressive neurologicdisease of the brain that leads to the irreversible loss of neurons anddementia. The clinical hallmarks of Alzheimer's disease are progressiveimpairment in memory, judgment, decision making, orientation to physicalsurroundings, and language.

“Neurodegenerative diseases” include i.a. Alzheimer disease, Parkinsondisease, Huntington disease, multiple sclerosis, amylotrophic lateralsclerosis (ALS), encephalitis or AIDS. [21]

Vicenin 2 is a flavonoid having a number of derivatizations. Vicenin 2or its synonyms Apigenin-6,8-di-C-glycoside,5,7,4′-Trihydroxyflavone-6,8-di-C-glucoside,5,7-Dihydroxy-2-(4-hydroxyphenyl)-6,8-bis[(2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6(hydroxymethyl)oxan-2-yl]chromen-4-one with the CAS Registry Number23666-13-9 is a flavonoid found in a number of plant species. Vicenin 2can be found i.a. in Anethum, Perilla species, Urtica species,Passiflora species, Camelia species, Cayaponia species, Cydonia species,Colocasia species, Desmodium species, Hordeum species, Origanum species,Ocimum species, Jatropha species, Parkinsonia species, Peperomiaspecies, Piheranthos species, Centaurea species, Indigo species, Bombaspecies, Lychnophera species, Asplenium species, Chinotto species,Citrus species, Viola species, Trigonella species, species belonging tothe Lamiacea, Labiatae and e.g. Rosemary, Peppermint, Thyme, Basil,Sage, Oregano, Lavandula, Nipponanthemum, Abrus, Viola, Santalum, Oryza,Scleropyrum, Tulsi, Centaurea, Indigofera, Bombax, Glinus, Lychnophoraand species belonging to Urticaceae, Rosales or Malpighiales.

The standard process to purify vicenin 2 out of plant material is basedon different chromatographic methods known by person skilled in the artand consequently, vicenin 2 may be identified by spectroscopy. Theseprocesses can be reviewed in several publications. [1,3]

So far the literature describes that vicenin 2 has an anti-cancer,anti-inflammatory and anti-nociceptive effect. [12, 13, 14]

“A plant extract comprising vicenin 2 or biologically active analoguethereof” is herein understood according to this invention to be anextract comprising vicenin 2 at a concentration which can be measured.The plant extract or preparation preferably comprises the activeingredient preferably at a concentration of at least 0.001%, 0.002%,0.005%, 0.008%, 0.010% or at least 0.015% or from 0.02% to 0.3%, morepreferably from 0.1% to 0.2% and preferably from 0.15% to 0.2%.

In some cases, the preparation may comprise the active ingredient at aconcentration of at least 0.001%, more preferably, at a concentration ofat least 0.002%, 0.005%, 0.008% or preferably at a concentration of atleast 0.010%, 0.015% or 0.02%.

In some cases, the preparation may comprise the active ingredient at aconcentration of at least 0.2%, more preferably, at a concentration ofat least 0.5%, 1.5%, 3.0% or most preferably at a concentration of atleast 2.0%, 3.0% or 5.0%.

As used herein “biologically active derivatives”, “biologically activeanalogues”, “functionally active derivatives” or “functionally activeanalogues”, which are used interchangeably herein, of vicenin 2 relateto structurally similar compounds to vicenin 2, e.g. flavonec-glycosides or flavone o-glycosides, in particular, apigenin7-O-β-glucuronide, apigenin-7-O-[β-glucuronosyl (1→2 β-glucuronide],luteolin 7-O-[β-glucuronosyl(1→2) β-glucuronide], luteolin7-O-β-glucuronide or scutellarein O-β-glucuronide (scutellarin), whichalso show effects beneficial for neurological function and brain health.These effects may include reversible acetylcholinesterase inhibition,blockade of gap junction hemichannels and reduction in TNF alpha. Thebeneficial effects to neurological function and brain health can bemeasured as shown in the examples.

There are several possibilities to investigate neurological effects,which are known to the skilled person. In vitro studies, like receptorbinding or enzyme inhibition studies, are standard methods known to theperson skilled in the art to identify activity and its mode of actions.This kind of test is shown in example 2. In addition it is possible toapply methods, which are known to the person skilled in the art andcommonly used within the preclinical development phase of pharmaceuticalagents in order to determine neurological activity when testing theacute neuroactive effects of substances on neuronal networks of murinecortex being grown on micro chips. By means of electrophysiologicalmulti-channel recording, electrical potential are compared to benchmarksto identify potential effects as well as side effects. This kind of testis shown in example 1. [15, 16, 17]

Furthermore, in vivo animal and in vivo human studies are used toconfirm the effect within the biological system. There are many wellestablished animal tests to investigate neurological abilities like forexample the forced swimming test. [1,8] Computer-based test for theassessment of working and short-term memory and selective attention aremany times used to assess cognitive function and progression incognitive disorders. [1,9] These kinds of studies are well known for theperson skilled in the art.

“The active ingredient” as used herein relates to vicenin 2 orfunctionally or biologically active derivative or analogue thereof.

In order to induce the beneficial neurological and brain health effects,the concentration of the active ingredient in the composition is 0.1 μgto 500 μg, preferably of 2.5 μg to 50 μg and more preferably of 5 μg to15 μg or 12 μg to 30 μg, most preferably about 24 μg vicenin 2.

The composition may be a plant preparation or plant extract, mostpreferably a liquid or powder extract obtained by extraction andcomprising vicenin 2.

The composition according to the present invention may be comprised in afunctional food product, dietary supplement or in a drug.

“A functional food product” according to this invention is understood tobe a food, beverage or infant formular product, which offers, inaddition, to nutritional value a health benefit, which supports andimproves health and wellbeing or helps to reduce the risk to develop adisease.

“A dietary supplement product” according to this invention is a foodproduct in form of a pill, tablet, capsule, pellet, globule, stickformulation, powder formulation, sachet formulation, powder or liquidform, which are meant to be taken by mouth, and contain substances likevitamins, minerals, foods, plant preparations, amino acids and areintended to supplement the usual intake of these substances via thenormal diet.

“A medicament/drug/medicine” according to this invention is anysubstance with the potential to prevent or cure disease or enhancephysical or mental welfare. If not stated otherwise the term “drug”,“medicine”, or “medicament” are used interchangeably herein and shallinclude but are not limited to all (A) articles, medicines andpreparations for internal or external use, and any substance or mixtureof substances intended to be used for diagnosis, cure, mitigation,treatment, or prevention of disease of either man or other animals; and(B) articles, medicines and preparations (other than food) intended toaffect the structure or any function of the body of man or otheranimals; and (C) articles intended for use as a component of any articlespecified in clause (A) and (B). The term “drug”, “medicine” or“medicament” shall include the complete formula of the preparationintended for use in either man or other animals containing one or more“agents”, “ingredients”, “compounds”, “substances” or “(chemical)compositions” as and in some other context also other pharmaceuticallyinactive excipients as fillers, disintegrants, lubricants, glidants,binders or ensuring easy transport, disintegration, disaggregation,dissolution and biological availability of the “drug”, “medicine”, or“medicament” at an intended target location within the body of man orother animals, e.g., at the skin, in the stomach or the intestine. Theterms “agent”, “compound” or “substance” are used interchangeably hereinand shall include, in a more particular context, but are not limited toall pharmacologically active agents, i.e. agents that induce a desiredbiological or pharmacological effect or are investigated or tested forthe capability of inducing such a possible pharmacological effect by themethods of the present invention.

“Pharmaceutically acceptable carrier” may include, but are not limitedto aqueous non-aqueous base solutions, suspensions, emulsions,microemulsions, micellar solutions, gels and ointments. Thepharmaceutically acceptable carrier may also contain ingredients thatinclude, but are not limited to, saline and aqueous electrolytesolutions; ionic and nonionic osmotic agents such as sodium chloride,potassium chloride, glycerol, and dextrose; pH adjusters and bufferssuch as salts of hydroxide, hydronium, phosphate, citrate, acetate,borate, and tromethamine; antioxidants such as salts, acids and/or basesof bisulfite, sulfite, metabisulfite, thiosulfite, ascorbic acid, acetylcysteine, cystein, glutathione, butylated hydroxyanisole, butylatedhydroxytoluene, tocopherols, and ascorbyl palmitate; surfactants such aslecithin, phospholipids, including but not limited tophosphatidylcholine, phosphatidylethanolamine and phosphatidylinositiol; poloxamers and poloxamines, polysorbates such as polysorbate80, polysorbate 60, and polysorbate 20, polyethers such as polyethyleneglycols and polypropylene glycols; polyvinyls such as polyvinyl alcoholand povidone; cellulose derivatives such as methylcellulose,hydroxypropyl cellulose, hydroxyethyl cellulose, carboxymethyl celluloseand hydroxypropyl methylcellulose and their salts; petroleum derivativessuch as mineral oil and white petrolatum; fats such as lanolin, peanutoil, palm oil, soybean oil; mono-, di-, and triglycerides; polymers ofacrylic acid such as carboxypolymethylene gel, and polysaccharides suchas dextrans, and glycosaminoglycans such as sodium hyaluronate. Suchpharmaceutically acceptable carriers may be preserved against bacterialcontamination using well-known preservatives, these include, but are notlimited to, benzalkonium chloride, ethylene diamine tetra-acetic acidand its salts, benzethonium chloride, chlorhexidine, chlorobutanol,methylparaben, thimerosal, and phenylethyl alcohol, or may be formulatedas a non-preserved formulation for either single or multiple use.

As used herein, the terms “treat” or “treatment” refer to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) an undesiredphysiological change or disorder, such as the development of a cognitivedysfunction, condition, disorder or disease. Beneficial or desiredclinical results include, but are not limited to, alleviation ofsymptoms, diminishment of extent of disease, stabilization (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, and remission (whetherpartial or total), whether detectable or undetectable. Those in need oftreatment include those already with the condition or disorder as wellas those prone to have the condition or disorder or those in which themanifestation of the condition or disorder is to be prevented.

These and other embodiments are disclosed and encompassed by thedescription and examples of the present invention. Further literatureconcerning any one of the materials, methods, uses and compounds to beemployed in accordance with the present invention may be retrieved frompublic libraries and databases, using for example electronic devices.For example the public database “Medline” or “Pubmed” may be utilized,which is hosted by the National Center for Biotechnology Informationand/or the National Library of Medicine at the National Institutes ofHealth. Further databases and web addresses, such as the virtual library“Martindale's center” are known to the person skilled in the art and canalso be obtained using internet search engines.

Several documents are cited throughout the text of this specification.The contents of all cited references (including literature references,issued patents, published patent applications as cited throughout thisapplication and manufacturer's specifications, instructions, etc.) arehereby expressly incorporated by reference; however, there is noadmission that any document cited is indeed prior art as to the presentinvention.

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific examples which are provided herein for purposes of illustrationonly and are not intended to limit the scope of the invention.

EXAMPLES

The examples which follow further illustrate the invention, but shouldnot be construed to limit the scope of the invention in any way.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of plant biology, chemistry,biochemistry, physiology and pharmacology which are within the skill ofthe art.

Example 1 Vicenin 2 and Perilla Extract Comprising Vicenin 2 and theirNeurological Efficacy Methods and Compounds

Purpose of this example was the evaluation of the acute neuroactiveeffects of vicenin 2 (named AUC-V) and of Perilla extract (named AUC-P)comprising and standardized on Vicenin 2 on the neuronal activity ofmurine frontal cortex networks in vitro by means of electrophysiologicalmulti-channel recordings. In this system, cells or tissues are growndirectly on the chip surface and communicate via chemical and electricalsignals. The MEA-neurochip allows the non-invasive, long-term, multisiterecording of electrical signal patterns of primary neuronal networks.Said MEA-neurochip technology enables the characterization of networkactivity pattern on the single-cell action-potential level and on thelevel of complex neuronal networks as the basic functional units. Thiscan also be used as a test system for investigating neuro-physiologicalproperties of compounds. Despite the complexity, the neurophysiologicalaction profiles of neuroactive compounds are sensitive and selective aswell as robust and stable, allowing a precise pharmacological“fingerprinting” and the creation of a database of information onwell-characterized neuroactive substances. The multiparametricdescription of the activity pattern changes caused by treatment of abrain-region-specific neuronal network is a sophisticated approach toquantify the complex effects of neuroactive agents, of unknowncompounds, and of complex mixtures such as biological extracts.Correlation with well-known neuroactive substances and their specificpattern, available in specific databased, provides novel insights intothe possible pharmacological and physiological mechanisms of modes ofaction of herbal preparations or isolated substances. [15, 16, 17]

In a first step the dose-effect curve of vicenin 2 (AUC-V) and the plantextract (AUC-P) were plotted by means of cumulatively increasing thesubstance concentration, so that the spectrum of activity of thesubstance is optimally covered with 9 concentrations. Concentration forVicenin 2 from 100 fg/ml, 10 pg/ml, 100 pg/ml, 1 ng/ml, 100 ng/ml, 1μg/ml, 10 μg/ml, 100 μg/ml and 300 μg/ml were used. Concentration forthe plant extract from 10 ng/ml, 100 ng/ml, 1 μg/ml, 10 μg/ml, 30 μg/ml,100 μg/ml, 200 μg/ml, 500 μg/ml, 1 mg/ml were used.

Subsequently, the measurements of the dose-effect curve of the testsubstances were repeated at least 10 times. The recorded electricalactivity patterns were characterized by 200 features and their changeswere statistically evaluated.

Afterwards, a further analysis was carried out through apattern-recognition analysis and through comparison with NeuroProofdatabase to determine relevant mechanisms involved in the frontal cortexactivity pattern induced changes by each substance.

Materials

The chemicals 5-fluoro-2′-deoxyuridine+uridine (FDU), and poly-D-lysinewere ordered from Sigma-Aldrich Chemical GmbH (Steinheim, Taufkirchen,Germany). DNase I (from bovine pancreas), and laminin were purchasedfrom Roche (Mannheim, Germany), fetal bovine serum from Pan Biotech GmbH(Aidenbach, Germany), and accutase from PAA (Germany). Horse serum andDulbecco's Modified Essential Medium (DMEM) were ordered from GIBCO BRL(Paisley, UK).

Microelectrode Array Neurochips

The microelectrode array neurochips (MEA neurochips) were provided bythe Center for Network Neuroscience (CNNS) at the University of NorthTexas. These 5×5 cm² glass chips have a central recording matrix with 64passive electrodes and indium tin oxide conductors. The hydrophobicinsulation material surface was activated by a brief butane flame pulsethrough a stainless steel mask. Thus, cell attachment on a confinedadhesive region (5 mm diameter centered on the electrode array) isensured. The activated surface regions were coated with poly-D-lysine(25 μg/ml; 30-70 kD) and laminin (16 μg/ml). Fabrication techniques andculture methods have been described previously.

Cell Culture

Frontal cortex tissue was harvested from embryonic day 15, or day 14chr:NMRI mice. After ethyl ether anesthesia, mice were sacrificed bycervical dislocation according to the German Animal Protection Act §4.Neuronal tissue was cultured including the use of DNase I (8000units/ml) and accutase (10 U/ml) for tissue dissociation. The tissue wasdissociated enzymatically with accutase and mechanically with transferpipettes. The cells were resuspended in DMEM 10/10 (10% horse and 10%fetal calf serum) at a density of 1.0×10⁶ cells/ml, and 400 μl wereseeded onto MEA surfaces. Cultures were incubated at 37° C. in a 10% CO₂atmosphere until ready for use, which usually is four weeks to threemonths after seeding. Culture media were replenished three times a weekwith DMEM containing 10% horse serum. Like in the tissue of origin,networks develop from a mixture of different types of postmitoticneurons and glial cells. The glial cells have important auxiliaryfunctions for the metabolism and for supplying the neurons with ions andnutrients. The developing co-cultures were treated with5-fluoro-2′-deoxyuridine (25 μM) and uridine (63 μM) for 48 h to preventfurther glial proliferation.

The cells growing directly on the neurochips emerge as natural neuronalnetworks. These are composed of a mixture of neurons and glial cellscomparable to the tissue of origin, whereas in interaction with theneurons, the glial cells fulfill various metabolism and transportfunctions. The neurons were coupled electrically to the neurochipelectrodes whereby the action potentials of the cells can be recordedand their amplitudes and the electrical activity pattern can beevaluated.

Activity starts after approximately three to four days in vitro in formof random spiking. Only after establishing a stable activity patternafter 4 weeks, the neuronal networks are employed in substance testing.For this study, cultures between 26 and 29 days in vitro were used.

Multichannel Recording

For extracellular recording, MEA neurochips were placed into sterilizedconstant-bath recording chambers and maintained at 37° C. Recordingswere made in DMEM/10% horse serum. The pH was maintained at 7.4 with acontinuous stream of filtered, humidified airflow with 10% CO₂. Sets ofpreamplifiers were positioned to either side of the recording chamber.Recording was performed with the multichannel acquisition processorsystem, a computer-controlled 64-channel amplifier system (Plexon, Inc.,Dallas, Tex., USA) providing programmable amplification, filtering,switching, and digital signal processing of microelectrode signals. Thetotal system gain used was 10K with a simultaneous 40 kHz sampling rate.The signals routinely recorded by these neurochips are located in arange of 15-1800 μV.

The multichannel signal acquisition system delivered single neuron spikedata. Spike identification and separation were accomplished with atemplate-matching algorithm in real time. This allowed the extracellularrecording of action potentials from a maximum of 256 neuronssimultaneously.

The action potentials, or “spikes”, were recorded in spike trains andare clustered in so-called bursts. Bursts were quantitatively describedvia direct spike train analysis using the program NeuroEXplorer (PlexonInc., Dallas, Tex., USA) and in-house programs. Bursts were defined bythe beginning and end of short spike invents. Maximum spike intervalsdefining the start of a burst were adjusted from 50 to 150 ms andmaximum intervals to end a burst from 100 to 300 ms.

Multiparametric Data Analysis

The high content analysis of the network activity patterns provided amultiparametric description characterizing the changes in fourcategories: general activity, burst structure, synchronicity andoscillatory behavior. The substance-specific activity changes werequantified by calculating for each stable activity phase after substanceapplication a total of 200 activity-describing spike train parametersfor these four categories below.

Statistical Analysis

Results are expressed as series means±SEM. The absolute parameters'distributions were tested for normality. The level of significance aftercompound application was assessed using Student's paired t-test.Significance between two substances or relative to a contained solvent(e.g. DMSO) was assessed using Student's unpaired t-test. P<0.05 wasconsidered statistically significant.

For direct comparability all parameters were normalized for eachexperiment and each experimental treatment with regard to thecorresponding values of the reference activity (native or after receptorblockade if applicable set to 100%).

For each experiment, the changing spike rate as a function of theconcentration was fitted to a one-sigmoidal or multiphasic-sigmoidaldose-response curve given by the equation:

$y = {y_{START} + \frac{y_{END} - y_{START}}{1 + 10^{{\lbrack{{\log {({EC}_{90})}} - {\log {(x)}}}\rbrack}^{*{nH}}}}}$

determining the values of the effective concentration causing 10, 50,and 90% effect (EC₁₀, EC₅₀, and EC₉₀) and of the slope (Hill coefficientnH; describes the slope of the curve: a high value corresponds to steepdecline which might correspond to functional neurotoxicity). In case ofmultiphasic response due to several mechanisms of action, the right termis repeatedly added for further phases.

Pattern Recognition and Classification

To clarify the mode of action of the test substance on the activity ofcortical networks these experiments were further analyzed using methodsof pattern recognition. For each stable concentration activity phase the200 spike train parameters were normalized by the native referenceactivity. These data records were computed for the test substances andthe reference substances.

Using a feature selection algorithm, on the basis of the referencesubstances, the 40 most descriptive parameters for all 200 spike trainparameters were selected. The rankings of activity features usingvarious score methods based on classification experiments and comparingtheir total correct predictions were calculated. In this manner, thebest results for a MDL (minimal description length) modified algorithmwere obtained. A training data set with these 40 spike train parameterswas established in the form of data records from the referencesubstances. An artificial neuronal network, multi layer feed forwardnetwork and back propagation algorithm without hidden units was thentrained. The respective data records of the four substances were allsubsequently classified. A classification against 105 substances in thedatabase was carried out.

Results:

Vicenin 2 showed an effect to induce a decrease of the cortical networkactivity with an EC₅₀ at 2 pg/ml and a maximum decrease to 80% of thenative activity with no affects on the burst structure, however on theoscillatory behaviour of the network. This means that the undesiredoverload of the cortical networks can be diminished by vicenin 2. Theseresults are shown in FIG. 1, which illustrates the concentrationdepending changes in the activity of the cortical network. It could beseen that vicenin 2 has an influence on the neuroactivity.

In addition, the extract comprising vicenin 2 has an effect to induce adecrease of the cortical network activity with an EC₅₀ at 90 ng/ml and200 μg/ml and a maximum decrease to 16% of the native activity with noaffects on the burst structure, however on the oscillatory behaviour ofthe network. These results are shown in FIG. 2, which illustrates theconcentration depending changes in the activity of the cortical network.It could be seen that the extract comprising vicenin 2 has an influenceon the neuroactivity.

In a second step, these activities were further evaluated by aclassification against positive controls.

Classifications for Vicenin 2 and the Perilla Extract

The classification was carried out against 105 substances using theelectrophysiological multi-channel recording of vicenin 2 as well as thePerilla extract.

TABLE 1 Classification of vicenin 2 (AUC-V) and the extract (AUC-P)against the NeuroProof database. Shown is a ranking of classificationresults, which means that x-% of all data records of this substance wereclassified as the respective substance in the left column; DScorresponds to the relative overall ranking. AUC-P, all 122 AUC-V, all143 reference # reference # Muscimol 25 Olanzapine 17 Eserine 24 Eserine15 DPDPE 19 Amisulpride 15 Acetaminophen 18 Enkephalin 14 Cortisol 14Sodium dodecyl sulfate 12 Amitriptyline 13 Atropinemethylbromide 12Amisulpride 12 Nicotine 12 Sodium propionate 12 Indatraline 12Quetiapine 11 Modafinil 12 Carbenoxolone 11 DPDPE 11

For example, it was possible to identify a sedative and anxiolyticactivity of vicenin 2 as well as of the Perilla extract by theclassification against Amisulpride, which is a known neuroleptica. Itwas also possible to identify a sedative and anxiolytic activity forPerilla extract by classification against Muscimol, which is a knownGABA receptor agonist.

Surprisingly, it was also possible to identify an enkephalin-likeactivity for vicenin 2. Enkephalins are endogenous ligands that bind tothe body's opioid receptors to modulate neuronal function, like learningor emotional behaviour, which can be altered by neurodegenerativedisorders. In Alzheimer disease, elevated enkephalin levels may reflectcompensatory or contribute to cognitive impairments.

Surprisingly, vicenin 2 a well as the extract comprising vicenin 2showed also an electrophysiological multi-channel recording comparablewith the pattern of the positive control eserine and nicotine. Theextract shows also an electrophysiological multi-channel recordingcomparable with the pattern of the positive control eserine. Eserine andanaloga are used in the treatment of Alzheimer disease as reversibleacetylcholinesterase inhibitors to improve short term memory. [6,7]

In addition, surprisingly vicenin 2 and the plant extract comprising thesame demonstrated an electrophysiological multi-channel recordingcomparable with the pattern of the positive control of carboxolone.Carbenoxolone blocks gap junction hemichannel of activated microglia andreduces their glutamate release and consequently diminishes cellautonomous neuronal death in neurodegenerative diseases. Therefore, gapjunction inhibitors are beneficial in treatment of neurodegenerativediseases. Carboxolone and analoga are used in the treatment of Alzheimerdisease. [10]

Further, both the standardized extract as well as the isolated vicenin 2demonstrated effects similar to amisulpride, which is a knownneuroleptic agent. In addition, the extract showed an effect toanti-depressive amitripthyline and the active ingredient vicenin 2 tothe anti-depressives and antipsychotics olanzapine and indatraline.Neuroleptika and antipsychotics are also used as companying therapy ofAlzheimer to increase mood and motivation and to decrease anxiety anddepression. This demonstrates further the effect of vicenin 2 and acomposition comprising it in neurological and/or brain function and/orin maintaining and/or improving neurological and/or brain functionand/or preventing, delaying onset, controlling and/or treating aneurological dysfunction, condition, disorder or disease.

Thus, example 1 surprisingly shows for the first time that vicenin 2 andan extract comprising the same (Perilla extract) can be used as abeneficial agent for neurological and brain functions or for thetreatment of a cognitive disorder. Cognitive functions include, memory,attention (concentration), alertness, learning, intelligence, language,problem solving capacity, coping with psychological stress or tension,anxiety and mood alterations.

Example 2 Perilla Extract Comprising and Standardized on Vicenin 2 hasthe Ability to Reduce TNFα

This study investigated the efficacy of Perilla extract comprisingvicenin 2 to contribute to reduce TNFα levels. The study was designed asan ex vivo study, where human whole blood samples were treated with LPS,a bacterial blend, to stimulate inflammation which leads to an increaseof the cytokine TNFα. Furthermore, the effect to reduce TNFα by Perillaextract was measured in comparison to a negative and positive control.Perilla extract demonstrated a dose-depending effect to reduce TNFα (seeFIG. 3).

Surprisingly, the extract as well as the isolated vicenin 2 demonstratedanti-inflammatory effects. Perilla extract demonstrated TNFα inhibitingefficacy within this ex vivo study showing a further beneficial effectin treatment if a cognitive disorder. In addition, TNFα has beenrecognized to be a gliatransmitter that regulates synaptic function inneural networks. Anti-TNF α treatment may thus lead to cognitiveimprovement. [8]

Even though anti-inflammatory agents have already been connected withAlzheimer disease, classical anti-inflammatory drugs, like NSAR, whichact via the prostaglandin pathway most probably do not work in Alzheimerdisease. Inflammation in the brain is mediated rather by activatedmicroglial cells having an increased metabolic activity resulting in theproduction of proteins, which may contribute to the invasion of plaques.In addition, they produce inflammatory markers, like cytokines.Therefore, inhibition of cytokines, like tumor necrose factor-alpha(TNFα), antioxidants and anti-inflammatory agents may also prevent theformation of amyloid beta, thus having a beneficial effect in Alzheimerdisease [8].

Alzheimer is a multiplex disease and each person reacts differently onpossible treatments. It is difficult to select the right treatment andtherefore, ingredients which combine several mode of actions arepreferred. However, before the present invention such agents having morethan one effective mode of actions were lacking. Surprisingly, thepresent inventors showed that vicenin 2 and functionally activederivatives thereof as well as plant extracts comprising the same havemore than one beneficial mode of actions for neurological and brainfunctions. This inventive agent may combine synergistically several modeof actions, i.e. at least blocking activity of gap junction hemichannelof activated microglia, inhibiting activity of tumor necrose factor αand inhibiting activity of acetylcholinesterase as well as havingneuroleptic, anti-depressive effects and enkephaline like effects, andprovides a new treatment possibility to maintain and improveneurological and/or brain function and to prevent and/or improvecognitive changes and to prevent and/or treat cognitive disorders,dementia including Alzheimer disease. The cognitive changes may be ageor stress related and even mild.

Isolated vicenin 2 and derivates thereof as well as plant extractscomprising the same may combine synergistically several mode of actionsbeing beneficial for maintenance and improvement of neurological andbrain functions and for preventing and treating a cognitive disorder,dementia such as Alzheimer disease. The combination to offer severalmode of actions within one agent is surprising and extremely beneficialas it contributes to a higher rate of success in the treatment of such amulti-complex and inter-individually disease like Alzheimer disease. Theinvention offers a highly effective beneficial agent and compositionwith a high compliance as individuals prefer to take one agent at onceinstead of many.

SUMMARY

Summarizing, vicenin 2 and biologically active derivatives thereof aswell as composition comprising the same demonstrated surprisinglybeneficial neurological efficacy.

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1. A method of maintaining and/or improving neurological and/or brainfunction and/or preventing, delaying onset, controlling and/or treatinga neurological dysfunction, condition, disorder or disease, the methodcomprising administering vicenin 2 or a biologically active analoguethereof as an active ingredient to a subject in need thereof.
 2. Themethod of claim 1, for maintaining and/or improving neurological and/orbrain function.
 3. The method of claim 1, wherein the neurologicaldysfunction, condition, disorder or disease is an age or stress relatedneurological dysfunction, anxiety, a cognitive disorder, depression ordementia.
 4. The method of claim 1, wherein the active ingredient isderived from a plant.
 5. The method of claim 4, wherein the activeingredient is comprised in a plant preparation enriched for the activeingredient.
 6. The method of claim 4, wherein the plant is selected ofone or more plants from a group consisting of a Anethum, Perilla,Urtica, Passiflora, Camelia, Cayaponia, Colocasia, Cydonia, Desmodium,Hordeum, Origanum, Ocimum, Jatropha, Parkinsonia, Peperomia,Piheranthos, Centaurea, Indigo, Bomba, Lychnophera, Asplenium, Chinotto,Citrus, Viola, Trigonella, Rosemary, Peppermint, Thyme, Basil, Sage,Oregano, Lavandula, Nipponanthemum, Abrus, Viola, Santalum, Oryza,Scleropyrum, Tulsi, Centaurea, Indigofera, Bombax, Glinus, Lychnophora,other species belonging to the Lamiacea, Labiatae, and Urticaceae,Rosales or Malpighiales or a combination thereof.
 7. The method of claim1, wherein the active ingredient is derived from a plant preparationselected from the group consisting of a leaf preparation, a fruitpreparation, a seed preparation, a stem preparation, a flowerpreparation, a bud preparation, a root preparation and a mixture ofdifferent parts of the plant.
 8. The method of claim 1, wherein theactive ingredient is an isolated vicenin 2 or biologically activeanalogue thereof obtained by isolation or chemical synthesis.
 9. Themethod of claim 1, wherein the neurological and/or brain function isselected from the group consisting of memory, attention, concentration,alertness, mental flexibility and/or speed, learning, intelligence,language skills, problem solving capacity, consciousness, coping withpsychological stress or tension, motivation, mobility, decision makingcapacity, reaction time and regulation of emotions.
 10. The method ofclaim 1, wherein the active ingredient is comprised in a composition andthe composition is a food product, a dietary supplement or a medicament.11. The method of claim 10, wherein the concentration of the activeingredient is from 0.1 μg to 500 μg.
 12. The method of claim 10, whereinthe composition is substantially free of flavonoids of other plants,other plant extracts or other plant flavonoids.
 13. The method of claim10, further comprising an agent other than vicenin 2 or the biologicallyactive analogue thereof capable of maintaining and/or improvingneurological and/or brain function and/or preventing, delaying onset,controlling and/or treating a neurological dysfunction, condition,disorder or disease.
 14. The method of claim 13, wherein the agent is alipid, a lipid containing omega-3-fatty acid, physiological active fattyacid, an antioxidant, an anti-inflammatory agent, a bulking agent, animmune system modulatory agent or a vaccine, an antibody, ametal-protein interaction attenuation agent, a plant preparation,curcumin, coenzym Q10, L-carnitine, zinc, epigallocatechingallate,thymol, p-cymere, vinpocetine, hyperzine A, phosphatidylserine, avitamin, alpha liponic acid, a TNF alpha inhibitor, a flavonoid, ananthocyanidin, a biflavonoid, a flavon, a flavonglycoside or acarboxylic acid.
 15. The method of claim 14, wherein the plantpreparation is selected from one or more extracts from a groupconsisting of an extract of Ginkgo biloba, Hypericum perforatum,Hyperzia serrata, Galanthus nivalis, Salvia officinalis, Panex ginseng,Lippia citriodora, Melissa officinalis, Passiflora incarnate, Passifloraedulis, Bacopa monniera, Zingiber officinalis, Leucojum aestrum,Concolulus pluricaulis, Centella asiatica, Emblica officinalis, CoptidisRhizoma, Salvia triloba, Piper nigrum, Trigonella foenum-graecum,Cimicifuga racemosa, Salvia miltiorrhiza, Rhodiola rosea, Habranthusjamesonii, Phycella herbertiana, Rhodophiala mendocina, Zephyranthesfilifolia, Stephania pierrei, Kaempfera parviflora, Stephania venosa,Crocus sativus, Salvia species, Bacopa monnieri, Centella asiatica,Ptychopetalum olacoides, Withania somnifera, Coptis chinensis, Mangiferaindica, Polygala caudata, Polygala tenuifolia, Halenia elliptica,Evolvilus alsinoides, Celastrus paniculatus, Clitoria ternatea, Curcumalonga, Acorus calamus, Terminalia chebula, Lycoris radiata, Magnoliaofficinalis, Biota orientalis, Codonopsis pilosula, Evodia rutaecarpa,Polygonum multiflorum, Aspalathus linearis, Cyclopia species, Adansoniadigitata, Sclerocarya birrea, Mangifera indica, Actinidia chinensisand/or Matricaria recutita or combinations thereof.
 16. The method ofclaim 13, wherein the agent is an acetylcholinesterase inhibitor, a NMDA(N-methyl-D-aspartate) receptor inhibitor, a non steroidalanti-rheumatic agent, a neuroleptic agent, a tricyclic antidepressant,an anti-psychotic agent, a gab junction inhibitor, a non selectivemonoaminooxidase inhibitor, an ABCC1 Transporter, an ADAM 10 protein,methylthioninium chloride, an antibiotic agent, an antiviral agent, agamma secretase inhibitor, a beta secretase inhibitor, an angiotensinreceptor antagonist (AT1 antagonist), a cannabinoid, allopregnanolone oran insulin sensitizer.
 17. A process of producing a preparation ofvicenin 2 or a biologically active analogue thereof as an activeingredient comprising the steps of: a) selecting a raw plant material,b) preparing the raw plant material, c) applying an extraction processusing aqueous solvent extraction and/or filtration techniques, d)determining the concentration of the active ingredient; and e) selectingthe preparation comprising the active ingredient at a concentration ofat least 0.01%.
 18. The process of claim 17, wherein the plant isselected from the group consisting of Anethum, Perilla, Urtica,Passiflora, Camelia, Cayaponia, Colocasia, Cydonia, Desmodium, Hordeum,Origanum, Ocimum, Jatropha, Parkinsonia, Peperomia, Piheranthos,Centaurea, Indigo, Bomba, Lychnophera, Asplenium, Chinotto, Citrus,Viola, Trigonella, Rosemary, Peppermint, Thyme, Basil, Sage, Oregano,Lavandula, Nipponanthemum, Abrus, Viola, Santalum, Oryza, Scleropyrum,Tulsi, Centaurea, Indigofera, Bombax, Glinus, Lychnophora, other speciesbelonging to the Lamiacea, Labiatae, and Urticaceae, Rosales orMalpighiales or a combination thereof.
 19. A preparation of the activeingredient obtained by the process of claim
 17. 20. (canceled) 21.(canceled)
 22. The method of claim 3, wherein the the dementia isAlzheimer disease.
 23. The method of claim 1, wherein the neurologicalor brain function is selected from the group consisting of memory,attention, concentration, alertness, mental flexibility and/or speed,learning, intelligence, language skills, problem solving capacity,consciousness, coping with psychological stress or tension, motivation,mobility, decision making capacity, reaction time and regulation ofemotions.
 24. A kit comprising vicenin 2 or a biologically activeanalogue thereof as an active ingredient and instructions foradministering said composition.
 25. The method of claim 1, wherein theneurological disorder is associated with autonomous neuronal death byblocking the gap junction hemichannel of activated microglia andreducing their glutamate release.
 26. The method of claim 25, whereinthe disorder is Alzheimer disease, Parkinson disease, Huntingtondisease, multiple sclerosis, amylotrophic lateral sclerosis (ALS),encephalitis or AIDS.
 27. The method of claim 25, wherein thecomposition comprises or is derived of a plant preparation comprisingthe active ingredient.
 28. The method of claim 27, wherein the plantpreparation is derived from a plant selected from a group consisting ofa Anethum, Perilla, Urtica, Passiflora, Camelia, Cayaponia, Colocasia,Cydonia, Desmodium, Hordeum, Origanum, Ocimum, Jatropha, Parkinsonia,Peperomia, Piheranthos, Centaurea, Indigo, Bomba, Lychnophera,Asplenium, Chinotto, Citrus, Viola, Trigonella, Rosemary, Peppermint,Thyme, Basil, Sage, Oregano, Lavandula, Nipponanthemum, Abrus, Viola,Santalum, Oryza, Scleropyrum, Tulsi, Centaurea, Indigofera, Bombax,Glinus, Lychnophora, other species belonging to the Lamiacea, Labiatae,and Urticaceae, Rosales or Malpighiales or a combination thereof and/oris plant preparation selected from the group consisting of a leafpreparation, a fruit preparation, a seed preparation, a stempreparation, a flower preparation, a bud preparation, a root preparationand a mixture of different parts of the plant.
 29. The method of claim1, wherein the active ingredient has more than one effect selected fromthe group consisting of reversible acetylcholinesterase inhibition,blockade of gap junction hemichannels, neuroleptic effect,anti-depressive effect, enkephaline like effect and reduction in TNFalpha.